Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse.

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. Although mice are widely applied to shed light on the mechanisms of cardiovascular disease, mouse models of AR, especially those induced by surgery, are still paucity. Here, a mouse model of AR was described in detail which is surgically induced by disruption of the aortic valves under high-resolution echocardiography. In accordance with regurgitated blood flow, AR mouse hearts present a distinctive and clinically relevant volume overload phenotype, which is characterized by eccentric hypertrophy and cardiac dysfunction, as evidenced by echocardiographic and invasive hemodynamic evaluation. Our proposal, in a reliable and reproducible manner, provides a practical guide on the establishment and assessment of a mouse model of AR for future studies on molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia-Reperfusion Injury by Modulating SIRT3-SOD2-mROS-Dependent Autophagy.

BACKGROUND: Ischaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short-term non-ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury. METHODS AND RESULTS: Transient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia-reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R-induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3-dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral-mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R-induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning. CONCLUSIONS: The present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self-defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway-dependent manner.

Difference in inflammation, atherosclerosis, and platelet activation between coronary artery aneurysm and coronary artery ectasia.

BACKGROUND: Coronary artery aneurysm (CAA) and coronary artery ectasia (CAE) may be two different types of coronary artery dilatation with unknown etiology. This study aimed to compare the differences between CAA and CAE and to investigate their pathogenesis and the necessity of antiplatelet therapy. METHODS: One hundred patients each with confirmed CAA, CAE, and normal coronary artery (NCA) from September 2017 to July 2019 were included. All patients completed examinations of the ankle-brachial index (ABI), pulse wave rate, and carotid ultrasonography; and were tested for routine blood, lipid, and immune parameters. Blood samples were collected 1 week after the withdrawal of antiplatelet drugs, and vascular inflammatory indexes, platelet activation indexes, thromboelastography, and the platelet aggregation rate were measured. Analysis of variance and the chi-square or Fisher exact test were used for statistical analysis. RESULTS: The perinuclear anti-neutrophil cytoplasmic antibody (ANCA), endothelial-1, matrix metalloproteinase-9, and tumor necrosis factor-α were significantly higher in CAE than in NCA, while cytoplasmic ANCA was appreciably higher in CAE than in CAA (P<0.05). Myeloperoxidase and growth/differentiation factor-15 were significantly higher in CAE than in CAA and NCA (P<0.05). ABI was significantly lower in CAA and CAE than in NCA (P<0.05), low-density lipoprotein/high-density lipoprotein was significantly higher in CAA than in NCA (P<0.05), and the detection rate of carotid artery thickening was significantly higher in CAA than in CAE and NCA (P<0.05). The Gensini and SYNTAX scores were significantly higher in CAA than in CAE (P<0.05). The percentages of CD62P and PAC-1 were higher in CAA and CAE than in NCA (P<0.05). The arachidonic acid aggregation rate in CAA and adenosine 5'-diphosphate aggregation rate in CAE were significantly higher than in NCA (P<0.05). The values of thrombin formation time and reaction time were significantly lower in CAE than in NCA (P<0.05), and the α angle was significantly higher in CAE than in NCA. CONCLUSIONS: CAE was closely related to inflammation, whereas CAA was closely related to atherosclerosis. Platelet activation was present in both diseases; therefore, antiplatelet therapy is recommended.

Mechanical stresses induce paracrine ß-2 microglobulin from cardiomyocytes to activate cardiac fibroblasts through epidermal growth factor receptor.

By employing a proteomic analysis on supernatant of mechanically stretched cardiomyocytes, we found that stretch induced a significantly high level of ß-2 microglobulin (ß2M), a non-glycosylated protein, which is related to inflammatory diseases but rarely known in cardiovascular diseases. The present data showed that serum ß2M level was increased in patients with hypertension and further increased in patients with chronic heart failure (HF) as compared with control group, and the high level of serum ß2M level correlated to cardiac dysfunction in these patients. In pressure overload mice model by transverse aortic constriction (TAC), ß2M levels in serum and heart tissue increased progressively in a time-dependent manner. Exogenous ß2M showed pro-fibrotic effects in cultured cardiac fibroblasts but few effects in cardiomyocytes. Adeno-associated virus 9 (AAV9)-mediated knockdown of ß2M significantly reduced cardiac ß2M level and inhibited myocardial fibrosis and cardiac dysfunction but not cardiac hypertrophy at 4 weeks after TAC. In vitro, mechanical stretch induced the rapid secretion of ß2M mainly from cardiomyocytes by activation of extracellular-regulated protein kinase (ERK). Conditional medium (CM) from mechanically stretched cardiomyocytes activated cultured cardiac fibroblasts, and the effect was partly abolished by CM from ß2M-knockdown cardiomyocytes. In vivo, knockdown of ß2M inhibited the increase in phosphorylation of epidermal growth factor receptor (EGFR) induced by TAC. In cultured cardiac fibroblasts, inhibition of EGFR significantly attenuated the ß2M-induced the activation of EGFR and pro-fibrotic responses. The present study suggests that ß2M is a paracrine pro-fibrotic mediator and associated with cardiac dysfunction in response to pressure overload.

Heat shock transcription factor 1 protects against pressure overload-induced cardiac fibrosis via Smad3.

Fibrotic cardiac muscle exhibits high stiffness and low compliance which are major risk factors of heart failure. Although heat shock transcription factor 1 (HSF1) was identified as an intrinsic cardioprotective factor, the role that HSF1 plays in cardiac fibrosis remains unclear. Our study aims to investigate the role of HSF1 in pressure overload-induced cardiac fibrosis and the underlying mechanism. HSF1 phosphorylation was significantly downregulated in transverse aortic constriction (TAC)-treated mouse hearts and mechanically stretched cardiac fibroblasts (cFBs). HSF1 transgenic (TG) mice, HSF1 deficient heterozygote (KO) mice, and their wild-type littermates were subjected to sham or TAC surgery for 4 weeks. HSF1 overexpression significantly attenuated pressure overload-induced cardiac fibrosis and dysfunction. Conversely, HSF1 KO mice showed deteriorated fibrotic response and cardiac dysfunction upon TAC. Moreover, we uncovered that overexpression of HSF1 protected against fibrotic response of cFBs to pressure overload. Mechanistically, we observed that the phosphorylation and the nuclear distribution of the Smad family member 3 (Smad3) were significantly decreased in HSF1-overexpressing mouse hearts, while being greatly increased in HSF1 KO mouse hearts upon TAC, compared to the control hearts, respectively. Similar alteration of Smad3 phosphorylation and nuclear distribution were found in isolated mouse cardiac fibroblasts and mechanically stretched cFBs. Constitutively active Smad3 blocked the anti-fibrotic effect of HSF1 in cFBs. Furthermore, we found a direct binding of phosphorylated HSF1 and Smad3, which can be suppressed by mechanical stress. In conclusion, the present study demonstrated for the first time that HSF1 acts as a novel negative regulator of cardiac fibrosis by blocking Smad3 activation. KEY MESSAGES: HSF1 activity is decreased in fibrotic hearts. HSF1 overexpression attenuates pressure overload-induced cardiac fibrosis and dysfunction. Deficiency of HSF1 deteriorates fibrotic response and cardiac dysfunction upon TAC. HSF1 inhibits phosphorylation and nuclear distribution of Smad3 via direct binding to Smad3. Active Smad3 blocks the anti-fibrotic effect of HSF1.

The effects of different angiotensin II type 1 receptor blockers on the regulation of the ACE-AngII-AT1 and ACE2-Ang(1-7)-Mas axes in pressure overload-induced cardiac remodeling in male mice.

Angiotensin II (AngII) type 1 receptor blockers (ARBs) have been effectively used in hypertension and cardiac remodeling. However, the differences among them are still unclear. We designed this study to examine and compare the effects of several ARBs widely used in clinics, including Olmesartan, Candesartan, Telmisartan, Losartan, Valsartan and Irbesartan, on the ACE-AngII-AT1 axis and the ACE2-Ang(1-7)-Mas axis during the development of cardiac remodeling after pressure overload. Although all of the six ARBs, attenuated the development of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) for 2 or 4weeks in the wild-type mice evaluated by echocardiography and hemodynamic measurements, the degree of attenuation by Olmesartan, Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally, the degree of downregulation of the ACE-AngII-AT1 axis and upregulation of the ACE2-Ang(1-7)-Mas axis was higher in response to Olmesartan, Candesartan and Losartan administration in vivo and in vitro. Moreover, in angiotensinogen-knockdown mice, TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan, Candesartan and Losartan but not by Telmisartan, Valsartan and Irbesartan administration. Furthermore, only Olmesartan and Candesartan could downregulate the ACE-AngII-AT1 axis and upregulate the ACE2-Ang(1-7)-Mas axis in vitro. Our data suggest that Olmesartan, Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II, possibly through upregulation of the expression of the ACE2-Ang(1-7)-Mas axis and downregulation of the expression of the ACE-AngII-AT1 axis. In contrast, Telmisartan, Valsartan and Irbesartan only played a role in the presence of AngII, and Losartan had no effect in the presence of AngII in vitro.

Mechanical Stress Regulates Endothelial Progenitor Cell Angiogenesis Through VEGF Receptor Endocytosis.

The clinical goal of cell-based treatment for chronic heart failure is to coordinately reconstitute the cardiomyocytes and associated circulation environment including coronary resistance arteries, arterioles, and capillary profiles.(1)) This goal can be possibly achieved by implementing multipotent adult stem cells. However, it remains a challenge to modify the capillary network in the decompensated heart. A mechanical stress model was used in this study to mimic the hemodynamic and hormonal states of the decompensated heart in vitro. The angiogenesis role of endothelial progenitor cells (EPCs) under stress has been well-recognized in vascular repair. We investigated the molecular mechanisms of EPCs in this model. We found that expression of vascular endothelial growth factor (VEGF) in EPCs was significantly decreased by mechanical stress, and this effect was accompanied by a decrease in angiogenesis in vitro. Interestingly, the defective angiogenesis can be reversed by upregulating the membrane VEGF receptor (VEGFR) endocytosis. An atypical protein kinase C (aPKC) inhibitor can promote the VEGFR internalization in EPCs and enhance the formation of vascular networks. Thus, the upregulation of VEGFR endocytosis in EPCs could be a potential therapy for the cell-based treatment of chronic heart failure by enhancing the cardiomyocytes.

Exenatide Reduces Tumor Necrosis Factor-α-induced Apoptosis in Cardiomyocytes by Alleviating Mitochondrial Dysfunction.

BACKGROUND: Tumor necrosis factor-α (TNF-α) plays an important role in progressive contractile dysfunction in several cardiac diseases. The cytotoxic effects of TNF-α are suggested to be partly mediated by reactive oxygen species (ROS)- and mitochondria-dependent apoptosis. Glucagon-like peptide-1 (GLP-1) or its analogue exhibits protective effects on the cardiovascular system. The objective of the study was to assess the effects of exenatide, a GLP-1 analogue, on oxidative stress, and apoptosis in TNF-α-treated cardiomyocytes in vitro. METHODS: Isolated neonatal rat cardiomyocytes were divided into three groups: Control group, with cells cultured in normal conditions without intervention; TNF-α group, with cells incubated with TNF-α (40 ng/ml) for 6, 12, or 24 h without pretreatment with exenatide; and exenatide group, with cells pretreated with exenatide (100 nmol/L) 30 mins before TNF-α (40 ng/ml) stimulation. We evaluated apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry, measured ROS production and mitochondrial membrane potential (MMP) by specific the fluorescent probes, and assessed the levels of proteins by Western blotting for all the groups. RESULTS: Exenatide pretreatment significantly reduced cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay at 12 h and 24 h. Also, exenatide inhibited excessive ROS production and maintained MMP. Furthermore, declined cytochrome-c release and cleaved caspase-3 expression and increased bcl-2 expression with concomitantly decreased Bax activation were observed in exenatide-pretreated cultures. CONCLUSION: These results suggested that exenatide exerts a protective effect on cardiomyocytes, preventing TNF-α-induced apoptosis; the anti-apoptotic effects may be associated with protection of mitochondrial function.

Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload-induced Cardiac Hypertrophy and Remodeling in Male Mice.

We previously showed that Qiliqiangxin (QL) capsules could ameliorate cardiac hypertrophy and remodeling in a mouse model of pressure overload. Here, we compared the effects of QL alone with those of QL combined with the following 3 types of antihypertensive drugs on cardiac remodeling and dysfunction induced by pressure overload for 4 weeks in mice: an angiotensin II type 1 receptor (AT1-R) blocker (ARB), an angiotensin-converting enzyme inhibitor (ACEI), and a ß-adrenergic receptor (ß-AR) blocker (BB). Adult male mice (C57B/L6) were subjected to either transverse aortic constriction or sham operation for 4 weeks, and the drugs (or saline) were orally administered through gastric tubes. Cardiac function and remodeling were evaluated through echocardiography, catheterization, histology, and analysis of hypertrophic gene expression. Cardiomyocyte apoptosis and autophagy, AT1-R and ß1-AR expression, and cell proliferation-related molecules were also examined. Although pressure overload-induced cardiac remodeling and dysfunction, hypertrophic gene reprogramming, AT1-R and ß1-AR expression, and ERK phosphorylation were significantly attenuated by QL alone, QL + ARB, QL + ACEI, and QL + BB, the attenuation was stronger in the combination treatment groups. Moreover, apoptosis was reduced to a larger extent by each combination treatment than by QL alone, whereas autophagy was more strongly attenuated by either QL + ARB or QL + ACEI. None of the treatments significantly upregulated ErbB2 or ErbB4 phosphorylation, and none significantly downregulated C/EBPß expression. Therefore, the effects of QL on chronic pressure overload-induced cardiac remodeling may be significantly increased when QL is combined with an ARB, an ACEI, or a BB.

CYP epoxygenase 2J2 prevents cardiac fibrosis by suppression of transmission of pro-inflammation from cardiomyocytes to macrophages.

Cytochrome P450 epoxygenase (CYP450)-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; but the underlying mechanism remains unclear. The present study aimed to elucidate how EETs regulated cardiac fibrosis in response to isoprenaline (Iso) or angiotensin (Ang) II. Cardiac-specific human CYP2J2 transgenic mice (Tr) and wild-type (WT) C57BL/6 littermates were infused with Iso- or Ang II. Two weeks after infusion, Tr mice showed more alleviative cardiac fibrosis and inflammation compared with WT mice. In vitro, we found Iso or Ang II induced nuclear transfer of NF-κB p65 and inflammatory cytokines expression in cardiomyocytes. Furthermore, inflammation response emerged in macrophages cultured in cardiomyocytes-conditioned medium. When pretreatment with 14,15-EET in cardiomyocytes, the inflammatory response was markedly suppressed and the transmission of inflammation from cardiomyocytes to macrophages was reduced. In conclusion, CYP2J2 and EETs prevent cardiac fibrosis and cardiac dysfunction by suppressing transmission of pro-inflammation from cardiomyocytes to macrophages in heart, suggesting that elevation of EETs level could be a potential strategy to prevent cardiac fibrosis.

ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer.

BACKGROUND: Recent studies have emphasized causative links between microRNAs (miRNAs) deregulation and tumor development. In hepatocellular carcinoma (HCC), more and more miRNAs were identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools. This study aimed to investigate the functional significance and regulatory mechanism of the miR-199a2/214 cluster in HCC progression. METHODS AND FINDINGS: In this study, we showed that miR-214, as well as miR-199a-3p and miR-199a-5p levels were significantly reduced in the majority of examined 23 HCC tissues and HepG2 and SMMC-7721 cell lines, compared with their nontumor counterparts. To further explore the role of miR-214 in hepatocarcinogenesis, we disclosed that the ER stress-induced pro-survival factor XBP-1 is a target of miR-214 by using western blot assay and luciferase reporter assay. Re-expression of miR-214 in HCC cell lines (HepG2 and SMMC-7721) inhibited proliferation and induced apoptosis. Furthermore, ectopic expression of miR-214 dramatically suppressed the ability of HCC cells to form colonies in vitro and to develop tumors in a subcutaneous xenotransplantation model of the BALB/c athymic nude mice. Moreover, reintroduction of XBP-1s attenuated miR-214-mediated suppression of HCC cells proliferation, colony and tumor formation. To further understand the mechanism of the miR-199a/214 cluster down-expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia-induced unfolded protein response (UPR) suppresses the expression of the miR-199a/214 cluster in HCC cells. By promoter analysis of the miR-199a2/214 gene, we conjectured NFκB as a potential negative regulator. We further found that UPR and LPS-induced NFκB activation suppressed miR-199a2/214 transcription, and this suppression was reversed by NFκB inhibition in HCC cells. CONCLUSIONS: Our study suggest that modulation of miR-214 levels may provide a new therapeutic approach for cancer treatment and revealed that UPR may offer a new explanation for why the miR-199a/214 cluster were down-regulated in the progression in HCC.